Phoenix dactylifera L. var. Ajwa, Hulwa, Ruthana, Sefri, and Luban. To determine changes in the fruit nutritive composition, primary metabolites, antioxidants, phenolic compounds and mineral profiles were analyzed in the fruits from non-fertilized and vermicompost-fertilized date palms. We also tested how changes in the fruit chemical compositions due to vermicompost fertilization affected their medicinal potentials. Applying vermicomposts generally increased primary metabolites, vitamins, and mineral content as well as the medicinal potential of the date palm fruits. This positive effect is possibly explained by the role of vermicomposts in improving soil health and fertility. Furthermore, clustering analyses and principal component analysis (PCA) indicated cultivar-specific responses. PCA analysis also revealed that the bioactivities of the date palm fruit extracts and their antioxidants tended to display correlated output values. One of the highly accumulated phenolic compounds, β-D-glucogallin, was extracted and purified from P. dactylifera L. var. Ajwa fruits and showed significant antioxidant, anticancer, antibacterial, antimutagenic, and antiprotozoal activities. Overall, applying vermicompost is an innovative approach to increase the nutritive quality and medicinal potential of date palm fruits.To meet the increased demand for phytochemicals, plant cultivation in soil amended with biofertilizers has been developed. Here, we aimed to use vermicompost as an environmentally safe biofertilizer to enhance the nutritive and medicinal value of five common cultivars of Saudi date palm; namely  Phoenix dactylifera L., Palmae) are the source of these fruits and are widely cultivated in the Middle East .From an ecological point of view, there is an increasing demand to apply eco-friendly approaches to improve productivity and food quality of agricultural crops. Applying bio-fertilizers containing beneficial microorganisms increased plant yield and helped in maintaining soil health and fertility . One widVermicompost is rich in humic acids and thereby provides a supportive environment for chemical reactions and microbial populations . In addiD-glucogallin. This molecule acts as an antioxidant and has antimicrobial activities  and mass spectrometry (HPLC/PDA/MS) were used to measure primary and secondary metabolites  in 12 cultivars of date palm fruits . One moltivities . β-D-gluications  and has ications .D-glucogallin, from P. dactylifera cv. Ajwa.In this study, we aimed to understand the potential effect of vermicompost fertilization on soil fertility and yield, nutritive quality and the medicinal potential of date palm fruits. Special emphasis was given to the accumulation of health-beneficial phytochemicals . Another aim was to evaluate the bioactivity of an isolated phenolic compound, β-The field study was performed during two successive growing seasons (2015 and 2016) in a private orchard located at Sakakah, Al Jawf region in the north of Saudi Arabia . Five cultivars of Saudi date palm  of 12 years of age were planted in sandy soil, in spaces of 6 × 6 m. Selected trees were healthy and as uniform as possible in age and growth. The number of leaves per bunch (9:1 ratio) was adjusted for each control and treated cultivars. No fertilizers were applied before the study. Date palm fruits were collected from the same trees in 2015 and 2016. In March of each year, 10 kg of vermicomposts were applied to the soil surface and then followed by irrigation. The experiment was conducted with three replicates for each treatment and three trees for each replicate; therefore, we used 12 trees for each cultivar. Soil water content was monitored by neutron moisture meter (neutron probe) and maintained at 60% of the soil water capacity. After 6 months, date palm fruits were collected from treated and untreated cultivars at the fully ripe (Tamar) stage. Washed date palm fruits were stored at -40°C before measurement of metabolic and mineral composition and extraction for biological activities evaluation. Results obtained for both harvests (2015 and 2016) were averaged.Soil physicochemical properties, pH, electrical conductivity (EC), organic matter (OM), total phenolic compounds and mineral nutrients were determined according to -1. Sugars were identified and quantified according to a calibration curve (1–10 mg 100 ml-1) of the corresponding standards of the same extracting solution.Mono, di- and polysaccharides in the date were measured by using HPLC following g and 4°C, the supernatants were passed through Millipore microfilters (0.2 μm pore size). Organic acids were detected by HPLC detection methods coupled with SUPELCOGELC-610H column and UV detector . Phosphoric acid  at a flow rate of 0.45 ml min-1 was used as a mobile phase.Quantitative determination of organic acids was performed according to g, the supernatant was resuspended in 5 ml of absolute chloroform. Immediately, the residue was re-extracted with 1 mL deionized water and centrifugation was repeated. The supernatant and pellet were suspended in absolute chloroform and then centrifuged at 8000 g for 10 min. The aqueous phase was filtered using Millipore microfilters (0.2 μM pore size). Amino acids were quantified using a Waters Acquity UPLC TQD system  coupled to a BEH amide column.One hundred mg of the dry fruit samples was homogenized in 5 ml of 80% aqueous ethanol  for 1 min at 5000 rpm . After c1.Fatty acid quantification was performed by gas chromatography according to -1. The extract was filtered, centrifuged and the resulted supernatant was evaporated under vacuum, then the residue was resuspended in methanol (HPLC grade). Baicalein (100 μg/ml) was used as an internal standard, and the concentration of the phenolic compounds was determined based on the corresponding standard.Phenolics and flavonoids were determined by using the method described in For determination of the total phenolics and flavonoids, 100 mg of the dry fruit samples was homogenized in 0.5 ml 80% aqueous ethanol. After centrifugation, the pellet was washed twice with 80% ethanol. A Folin–Ciocalteu assay was used for total phenolic content quantification , with gaContents of ascorbate, tocopherols, and carotenoids were determined in date palm fruits by HPLC. The separation and detection processes were done with UV and/or fluorescence detectors . Tocophe3/H2O solution (5:1 v/v) in an oven according to One hundred mg of the dry fruit samples were digested in HNOStreptococcus spp., Serratia marcescens, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa) obtained from the microbial culture collection of the Department of Clinical Laboratory Sciences, Al Jouf University, Saudi Arabia were used as test strains. About 150 μl of the bacterial suspension was spread on Muller Hinton agar and nutrient agar media. The same solvent employed to extract the date palm fruits was used as a negative control. Amoxicillin (30 μg/disc), gentamycin (30 μg/disc), and streptomycin (30 μg/disc) were used as positive reference standards. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined for the strains sensitive to the fruit extract in the assay. Time-kill studies were performed for S. aureus in McCartney bottles using a method based on the European Standard quantitative suspension test.The disc diffusion method was used to evaluate the bacterial potential of the date palm fruit extracts . The cliD-glucogallin. The two strains (TA98 and TA100) of Salmonella typhimurium were used for this test. Sodium azide (NaN3) and mutagen 4-nitro-o-phenylenediamine (NPD) were used as positive controls. Toxicity of β-D-glucogallin was checked against all the used mutagens. The experiments were conducted in pre-incubation and co-incubation modes. The inhibition % of mutagenic activity was calculated as described by D-glucogallin and β-D-glucogallin alone were used for the calculations.An Ames assay  was usedLeishmania donovani and Trypanosoma cruzi was conducted according to the protocols established by 8 parasites per mL, and 90 μL were added to each well of the 96-well plates. Then, 10 μL of β-D-glucogallin and standard compound (Benznidazole and Miltefosine) solutions were added to the wells. Amastigotes of L. donovani were incubated with AlamarBlueTM for 3 days, and the viability was evaluated by a fluorescence scanner. Viability measurements of trypomastigotes of T. cruzi and L. donovani were conducted using rat skeletal myoblasts and seeded in the microtiter plates using chlorophenol red-β-D-galactopyranoside (CPRG)-Nonidet substrate. After 4 days of incubation at 37°C and under 5% CO2 environment, the developed color reaction was read at 540 nm. The IC50 values were calculated for both species.The vitro anti-leishmanial assay against the amastigotes of D-glucogallin  was mixed with the samples or with the standard (trolox) solution in micro-titration plates for 30 min and measured at 593 nm  was assayed by extracting date palm fruits in 80% ice-cold ethanol in liquid nitrogen. FRAP reagent  dissolved in ethanol were used. Cell lines were grown in Dulbecco’s Modified Eagle Medium (DMEM)  at 37°C and 5% CO2. The cell lines were suspended and seeded in DMEM. Incubation of the cell lines with extracts, β-D-glucogallin and ethanol as a negative control was continued until a confluent growth was achieved. Then, the cells were collected by trypsinization in trypsin solution (0.25% trypsin w/v) and the vital cells were determined by the CellTiter-Blue reagent  (Human cancer cell lines  were used in this study. Ethanol extracts of the tested date palm fruits (2 mg/mL) as well as β- States) .D-glucogallin, 250 g of P. dactylifera cv. Ajwa date fruits, which showed the highest concentration of β-D-glucogallin, was collected and extracted in ethanol for up to 6 h. Then, the filtrate was concentrated under vacuum. Dried ethanol extract was eluted by using gradients of heptane:ethanol (9:1 to 0:1) as a mobile phase to yield seven fractions depending upon thin layer chromatography (TLC) analyses. The abundant fraction, was evaporated under reduced pressure at 45°C to remove the solvent and then subjected to column chromatography  on a silica gel column. The mobile phase (dichloromethane:water) with gradient from 10:0 to 3:10 was chosen for elution depending upon TLC analyses to give 5 sub-fractions (F1-1 to F1-5). Sub-fraction 2 (F5-2) (3.14 g) showed the highest elongation activity by using coleoptiles bioassay. In this assay, 5 mm of wheat coleoptiles were placed in test tubes containing the extracts and controlled tubes were filled with water. Test tubes were moved to growth cabinet for 24 h, and then the increases in collectibles length were recorded. TLC analyses of F1-3 showed the presence of mixture of three compounds, and one of them was very low, the major one was purified by subjecting to Sephadex LH-20 column and 104.5 mg of the purified compound was yielded. The preliminary bioactivity test of the isolated pure compound indicated its phytotoxicity.To isolate β-1H, and 13C NMR spectra were used to identify the pure isolated phenolic compound β-D-glucogallin. Samples were dissolved in acetone and the internal standard tetramethylsilane (TMS) was used. The compound in F1-3 was identified based on spectroscopic analyses and comparison of 1H and 13C NMR data with previous literature values  was applied by using SPSS 16.0 . PCA analysis was performed by using XLSTAT . All parameters were subjected to Pearson distance metric cluster analysis by using the MultiExperiment Viewer (MeV) TM4 software package .One-way analysis of variance . For instance, malic acid is one of the abundant organic acids in date fruits and significantly contributes to the characteristic fruity, smooth and sour taste of the fruits . OrganicIn date palm fruits, lipid content represents about 10% of dry mass . For insA high content of total phenolic and flavonoids was recorded in non-treated date palm fruits in the range of 0.3–4 mg/g DW . BecauseDate palm fruits are good sources for vitamins such as Vit-C (L-ascorbic acid), Vit-A (carotene), Vit-B1 (thiamine), and Vit-B2 (riboflavin) . Beside In general, significant amounts of minerals in date palm fruits were observed, and they were significantly induced by vermicompost treatment . In partStreptococcus spp. and E. coli. Antioxidant and anticancer capacities of the ethanol extracts of date palm fruits could be attributed to the availability of antioxidants such as phenolics, flavonoids, steroids, tocopherols, and ascorbate  of β-D-glucogallin under all applied concentrations was recorded. More specifically, β-D-glucogallin showed higher anti-mutagenic activity at a concentration of 2500 μg/plate against S. typhimurium TA98. For both tested bacterial strains, mutagenicity decreased from 45 to 5 for TA98 and from 36 to 12 for TA100. The induced anti-mutagenic activity of β-D-glucogallin could be attributed to its powerful radical scavenger activity. β-D-glucogallin reduces the mutagenicity caused by indirect acting mutagen cyclophosphamide by 45 and 36%, respectively, in the strains TA98 and TA100 at the highest tested dose (2500 g/plate), which proves the strong anti-mutagenic activity. It also had strong and effective anti-mutagenicity against cyclophosphamide. With respect to the antiprotozoal activity, β-D-glucogallin showed high activity against T. cruzi and L. donovani  were also recorded for β-D-glucogallin (Based on the promising antimicrobial activity, the anti-mutagenic activity was also investigated by the Ames test against the indirectly acting mutagen cyclophosphamide . It has donovani . IC50 vacogallin . DPPH raP. dactylifera cv. Ajwa date fruit extract was a good source of the natural bioactive compound β-D-glucogallin, which showed significant antioxidant activity as well as promising action against microbial and protozoal growth.The findings of the present work suggested that fertilizing palm date fruits with vermicompost is a promising eco-friendly approach to improve date palm fruit yield, nutritive quality and biological activities. Vermicompost improved soil health and fertility by increasing nutrients, plant growth regulators and humic acids. Improved soil fertility could explain the high health-beneficial value of the produced date palm fruits as indicated by the elevated levels of primary metabolites, minerals, vitamins, essential amino acids, unsaturated FA and antioxidants. Consequently, increased amounts of compounds that are beneficial to health enhanced the medicinal value of date fruit ethanol extracts. Our results also signify that SAJ, SS, SH, WH, HA, and HAE set-up the experiments. HAE, SS, and HM did the analyses and HAE, HA, and WH wrote the manuscript. All authors revised and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Escherichia coli (E. coli) and Gram-positive bacterium Bacillus subtilis (B. subtilis). The average diameter of the bacteriostasis loops against both E. coli and B. subtilis becomes larger with an increasing silver loading amount.This work provides a universal, fast, simple, and environmentally-friendly cold plasma method for synthesizing Ag NPs on heat-sensitive materials at atmospheric pressure.Cotton-fabric-supported silver nanoparticles (Ag NPs) have aroused great attention due to their remarkable physical and chemical properties and excellent broad-spectrum antibacterial performance.In this work, a surface dielectric barrier discharge (DBD) plasma method is developed and employed to prepare cotton fabric supported Ag NPs (Ag/cotton) for the first time. UV-Vis and X-ray photoelectron spectroscopy (XPS) results confirm the formation of Ag NPs. TEM images show that the size of Ag NPs is in the range 4.8–5.3 nm. Heat-sensitive cotton fabrics are not destroyed by surface DBD plasma according to FTIR and XRDresults. Wash fastness of the Ag/cotton samples is investigated using ultrasonic treatment for 30 min and it is shown that the Ag NPs possess good adhesion to the cotton fabric according to UV-Vis spectra. Antibacterial activity of the Ag/cotton samples shows that obvious bacteriostasis loops are observed around the samples with the appearance of both Gram-negative bacterium  Cotton fabrics are highly popular due to their excellent properties such as comfortability, air permeability, flexibility, bio-degradable ability, and good absorption of water. However, they provide ideal conditions  for bacteria and fungi to grow due to their high specific surface areas. In cotton fabrics, therefore, pathogens breed quickly, send out an unpleasant odor, and lead to textile decoloration and value shrinking, which accelerates the cross-spread of infection and aggravates human health. To solve these problems, great efforts have been devoted to modifying cotton fabrics by adding various antibacterial chemicals  to improve their antibacterial activity ,2,3,4,5.3 concentration. The loomstate- and scoured-cotton-fabric-supported Ag NPs exhibited excellent durability and antibacterial activity. As mentioned above, both the exsitu method and insitu method have prepared cotton-fabric-supported Ag NPs with high antibacterial activity and durability. However, excess chemical reagents are generally required to be served as reducing agents. Therefore, more attention has been paid to environmentally-friendly preparation methods, such as the bio-synthesis method [According to the reduction process of silver ions into Ag NPs, the methods for preparing cotton-fabric-supported silver nanocomposites can be categorized into an exsitu reduction method and an insitu reduction method. With regards to theexsitu reduction method, an aqueous colloidal solution of Ag NPs is first prepared and then is supported on cotton fabrics by functional groups ,8. For es method , photo-rs method , and cols method .E. coli and B. subtilis. Compared to low-pressure (LP) cold plasma, atmospheric-pressure (AP) cold plasma, which does not require high-cost and sophisticated vacuum equipment, is more attractive. However, since the electron temperature of AP cold plasma is generally low at atmospheric pressure, hydrogen-containing species  should be used as the reducing agents [Cold plasma is characterized by a high electron temperature and low gas temperature (which can be close to room temperature). It has been proven to be an environmentally-friendly and fast method for reducing metal ions to synthesize supported metal catalysts due to its non-thermal equilibrium properties ,19. The g agents ,27,28. CE. coli and the Gram-positive bacterium B. subtilis.Surface DBD is one important type of DBD discharge in which the discharges occur between the dielectric and discharge electrodes. It has been widely used in aerodynamic flow control , seed tr−2) were cut into small pieces (2.5 × 2.5 cm2) and cleaned by Triton X-100 at 60 °C for 1 h. Silver nitrate (AgNO3) and polyvinyl pyrrolidone  were purchased from Kemiou Chemical Reagent Co., Ltd. . Ag/cotton samples were prepared by impregnation, which was followed by cold plasma treatment. Typically, a piece of cotton fabric was put into the AgNO3 aqueous solution and PVP and kept in the dark for 2 h. Then, the wet samples were centrifuged at 6000 rpm for 10 min to remove the excess AgNO3. Afterwards, the samples were flattened and put into asurface DBD reactor.Prior to preparing the cotton-fabric-supported silver (Ag/cotton) samples, 100% plain-woven cotton fabrics (320 g·m2 in area. The induction electrode was a 7.6 × 4.2 cm2 rectangle. A mixture of high purity Ar and H2 (>99.999%) was adopted as the working gas and the generated active hydrogen species  were used to reduce the silver ions into Ag NPs [−1 and the gas ratio of Ar to H2 was 1:1. A CTP-2000K power supply  was employed to ignite the surface DBD electrode system to generate AP cold plasma. The discharge voltage was a 5.7 kV peak-to-peak sinusoidal high voltage and the discharge frequency was kept at 10.4 kHz. The Ag/cotton samples were placed below the surface DBD electrode system at a 4 mm distance. The treatment was performed three times, taking 2 min for each treatment. After surface DBD plasma treatment, the as-prepared Ag/cotton samples were dried at 120 °C for 2 h under vacuum in the dark. Then, they were rinsed with deionized water to get rid of the residual water-soluble AgNO3 salt and dried under vacuum for another 2 h. Various AgNO3 concentrations  were adopted, while the PVP concentration was kept constant at 10 mg·ml−1. Accordingly, the Ag/cotton samples prepared by surface DBD plasma were marked as Ag/Cotton-1, Ag/Cotton-2, Ag/Cotton-5, and Ag/Cotton-10, respectively.o Ag NPs ,12. The −1 with a resolution of 4 cm−1 by accumulating 32 scans. UV-Vis diffuse reflectance spectra (DRS) of the samples were collected for the spectral range 200–800 nm using a Hitachi U-3900 UV-Vis spectrometer  with BaSO4 as a reference. XRD data of the samples were examined via a Dandong Haoyuan DX-2700 X-ray diffractometer  using CuKα1 radiation (λ = 1.54178 Å). The morphology of the samples was examined using a Zeiss Sigma500 SEM  at a 5 kV accelerating voltage. Prior to SEM investigation, the cotton fabric samples were coated with a thin film of gold by sputtering. The SEM images were recorded at magnifications of 5000× and 10,000×. TEM and high resolution TEM (HRTEM) experiments were carried out using a FEI Tecnai G2 F20 S-Twin TEM . Prior to testing, the samples were cut into pieces and placed in ethanol with ultrasonic dispersion for 120 min. Then, a few drops of the suspension were supported on an ultra-thin carbon film and dried in air at room temperature. The sizes and size distribution of the Ag NPs were obtained by calculating sizes for more than 300 Ag NPs.X-ray photoelectron spectroscopy (XPS) experiments were performed on a Thermo Fisher Scientific K-Alpha XPS spectrometer  to investigate the valence state of the silver element. The binding energies were calibrated using a C1s peak at 284.6 eV as the reference. Infrared spectra of the samples were recorded on a Thermo Scientific Nicolet 6700 Fourier Transform Infrared Spectrometer  using attenuated total reflection in the region of 6000–600 cm2) was immersed in a Pyrex beaker with 30 mL of deionized water. Then, the beaker was put into a KQ2200DB digital control ultrasonic generator  with a frequency of 40 kHz and an ultrasonic input power of 100 W. Ultrasonic leaching was performed for 30 min at a 30 °C sonication bath temperature. Then, the samples were dried under vacuum at 120 °C for 2 h. The wash fastness of the samples was tested by comparing their UV-Vis DRS spectra before and after ultrasonic leaching treatment.Wash fastness of the Ag/cotton samples was estimated by ultrasonic leaching method. Typically, a piece of Ag/cotton sample  was evaluated by the disk diffusion method and dilution method of plate counting. The process was similar to that used in a previous work [−1 beef powder, 1.5 g∙L−1 soluble starch, 17.5 g∙L−1 acid hydrolyzed complex protein, and 17 g∙L−1 agar), deionized water, and the petridish were autoclaved prior to use. To compare antibacterial activity, blank cotton fabric and the Ag/cotton samples were cut into pieces of equal size (1.0 × 1.0 cm2). The E. coli and B. subtilis bacteria were cultured with MH agar medium in a constant temperature incubator at 37 °C for 8 h, respectively. Afterwards, the bacteria cultures were diluted  with deionized water and MH agar, and the inoculums were evenly spread over the petridish, respectively. Lastly, the samples were planted onto the agar plates and incubated at 37 °C for 24 h to test their antibacterial activity by examining the diameter of the growth-inhibition zone.The antibacterial activity of the Ag/Cotton samples against the Gram-negative bacterium ous work . Mueller3 can be reduced to metallic silver by surface DBD plasma. Moreover, by increasing the loading amount of silver, the color of the Ag/cotton samples changes from light yellow to brownish yellow, indicating that more Ag NPs are formed at a high silver loading amount. Similar phenomena were also observed in the preparation of Ag/cotton samples by DC glow discharge cold plasma at low pressure [pressure .To further confirm the reduction ability of the surface DBD plasma, an XPS spectrum of Ag3d in Ag/Cotton-5 was measured, as is presented in −1 [−1 for the samples corresponds to the stretching vibration band of the O-H groups. The medium bands at 2800–3000 cm−1 may be ascribed to the symmetric and asymmetric stretching vibrations of the C-H groups in the cellulose samples [−1 can be assigned to adsorbed water in the samples. The peaks at 1484 cm−1 and 1385 cm−1 correspond to the wagging and bending of the C-H functional groups in cellulose, respectively. All of the Ag/cotton samples prepared by surface DBD plasma exhibited similar FTIR spectra to the blank cotton and no new peaks were able to be detected from the FTIR spectra of the Ag/cotton samples. This indicates that surface DBD plasma has no obvious influence on the structure of the cotton fabric support. The nondestructive property of surface DBD plasma is attributed to the fact that the heat-sensitive cotton fabric was not closely contacted with the discharge zone but below it at a 4 mm distance. Hence, the generated active hydrogen species in surface DBD plasma can reduce the silver ions into metallic silver without destroying the cotton fabric.As shown in −1 . The bro samples . The banAs illustrated in To determine the influence of silver deposition on the morphology of the cotton fabric and the distribution of the Ag NPs, typical SEM images of Ag/Cotton-1, Ag/Cotton-10, and the blank cotton sample wereobtained, as shown in DAg) was obtained by measuring more than 300 Ag NPs, and the histograms of size distribution of the AgNPs are also illustrated in DAg in Ag/Cotton-1 and Ag/Cotton-10 were found to be 4.8 ± 1.7 and 5.3 ± 1.9 nm, respectively, demonstrating a slight increase in the size of the Ag NPs at a higher silver loading amount. The size of the Ag NPs detected by TEM was much smaller than that obtained by SEM, indicating the tendency of aggregation of Ag NPs. The Ag NPs were thought to be aggregated after cold plasma reduction due to the hydrophilic property of cellulose [Fm3m, a = 4.086Å, JCPDS card, file no. 65-2871). This is in line with the XPS and UV-Vis DRS results, revealing that metallic Ag NPs with high crystalline quality are obtained by surface DBD plasma treatment.To clearly disclose the distribution of Ag NPs in the Ag/cotton samples, TEM images of Ag/Cotton-1 and Ag/Cotton-10 were obtained, as shown in ellulose . The corWash fastness of the Ag/cotton samples was estimated using ultrasonic treatment. UV-Vis DRS spectra of the Ag/cotton samples as-synthesized and after 30 min of ultrasonic treatment are presented in E. coli and Gram-positive bacterium B. subtilis with the same concentration were employed to evaluate the antibacterial effect of the Ag/cotton samples, and the results are illustrated in E. coli and B. subtilis. However, obvious bacteriostasis loops can be observed around the Ag/cotton samples with the appearance of E. coli or B. subtilis. These findings indicate that the surface DBD plasma is efficient for preparing Ag/cotton samples with high antibacterial activity, which may be ascribed to the small size and high distribution of Ag NPs. The average diameters of the bacteriostatic loops for Ag/Cotton-1, Ag/Cotton-2, Ag/Cotton-5, and Ag/Cotton-10 against E. coli were found to be 1.38, 1.39, 1.45, and 1.63 cm, respectively. By contrast, the average diameters of the bacteriostatic loops for Ag/Cotton-1, Ag/Cotton-2, Ag/Cotton-5 and Ag/Cotton-10 against B. subtilis were 1.85, 1.86, 2.05, and 2.18 cm, respectively. The average diameters of the bacteriostasis loops against both E. coli and B. subtilis became larger with an increase in the silver loading amount, demonstrating that Ag/cotton samples with a higher silver concentration possess higher antibacterial activity. Moreover, the diameters of the bacteriostasis loops against B. subtilis for the same Ag/cotton samples were larger those that against E. coli, suggesting that the Ag/cotton samples exhibit higher antibacterial activity against Gram-positive bacterium B. subtilis than Gram-negative bacterium E. coli, which is in line with the results of Li et al. [The Gram-negative bacterium i et al. . This mai et al. .E. coli and the Gram-positive bacterium B. subtilis. This work provides a universal, fast, simple, and environmentally-friendly cold plasma method for synthesizing Ag NPs on heat-sensitive materials at atmospheric pressure.Cotton-fabric-supported Ag NPs (Ag/cotton) have been successfully prepared in this work using an environmentally-friendly and fast surface DBD plasma method for the first time. No additional reducing chemicals except the active hydrogen species generated in cold plasma served as the reducing agents. The color of the samples changed from colorless to yellow after surface DBD plasma treatment and the color changed from light yellow to brownish yellow with an increase in the loading amount of silver. Moreover, SPR peaks and the Ag3d XPS results further confirm that the Ag species mainly exist in the metallic state. TEM images show that the Ag NPs are uniformly distributed on the fiber surface and that the size of the Ag NPs is in the range 4.8 to 5.3 nm. FTIR and XRD results demonstrate that heat-sensitive cotton fabrics are not destroyed by surface DBD plasma, which may be due to the fact that the cotton fabric is not in close contact with the discharge zone during the treatment but below it at a 4 mm distance. Wash fastness estimated using ultrasonic treatment revealed that the Ag NPs in the Ag/cotton samples possess high adhesion ability to the cotton fabric. The Ag/cotton samples exhibit high antibacterial activity for both the Gram-negative bacterium


Low protein expression of E-cadherin in oral squamous cell carcinoma (OSCC) has been associated with clinical and histopathological traits such as metastases, recurrence, low survival and poor tumor differentiation, and it is considered a high-risk marker of malignancy. However, it is still unknown whether low expression of E-cadherin is also present at the mRNA level in OSCC cases. Objective: The aim of this study was to compare E-cadherin mRNA expression in OSCC patients and controls and to correlate the expression with clinical and prospective characteristics.Forty patients and 40 controls were enrolled. E-cadherin mRNA expression was evaluated by quantitative real-time polymerase chain reaction using TaqMan probes.p<0.001). Whereas no significant association between clinical parameters and E-cadherin expression levels was observed, we noted lower E-cadherin expression levels in patients with positive lymph node metastasis.E-cadherin mRNA expression was significantly decreased in OSCC patients compared to that of controls (E-cadherin mRNA expression was markedly diminished in OSCC, in agreement with previous results that examined E-cadherin expression at the protein level. E-cadherin is downregulated in the early clinical stages of OSCC, and its mRNA levels do not change significantly in the advanced stages, suggesting that there is limited usefulness of this parameter for predicting disease progression. Key words:Oral cancer, E-Cadherin, gene expression. Global demographic transitions suggest that there will be an increase in cancer burden over the next several decades, particularly in middle- and low-income countries . In 2012Oral squamous cell carcinoma (OSCC) is the most common histological cancer type of the oral cavity , and it In the advanced stages, neoplastic cells undergo a dedifferentiation process, which is followed by a loss of intercellular adherence. Intercellular adhesion is primarily maintained by cadherin adhesion molecules, and specifically by E-cadherin in the epithelial tissue . E-cadheAccordingly, low expression of E-cadherin in OSCC has been associated with clinical and histopathological features of malignancy, such as metastasis, recurrence, low survival and poor tumor differentiation -13, and A total of 40 cases of primary OSCC were diagnosed over a period of 3 years (2012-2013)  at the Instituto Jaliscience de Cancerología ; no anticancer therapy had been initiated in any of these cases. The 40 control samples were obtained from patients  who visited the maxillofacial surgery service at the Hospital Civil Nuevo  for tooth extraction with prosthetic and orthodontic indications. For each patient, data regarding the following clinico-pathological factors were collected: gender, age, smoker status, alcohol consumption, TNM stage, recurrence, persistence and date of death. The exclusion criteria for both groups were having or having had any type of cancer other than OSCC, being diagnosed with an autoimmune disease that affected the oral cavity, such as pemphigus vulgaris or lichen planus, or not signing the informed consent. Protocol was reviewed and accepted by Universidad de Guadalajara biosecurity and etic committee number CI-02112-Clinical and prospective dataThe stage of the disease was evaluated at the Surgical Head and Neck Oncology Department of the Jaliscience Cancer Institute, and each tumor was analyzed clinically and by computerized tomography. The disease stages were then determined according to the TNM system (AJCC) .Due to the finding that the diameter of the tumor and invasion into lymphatic nodes are regarded as important clinical markers involved in prognosis, the lymphatic nodes were considered positive if patients presented with metastasis in one or more lymphatic nodes. T stage and total TNM scores were assigned in accordance with the National Cancer Institute guidelines, and each case was determined to be early- or advanced-stage disease  . Accordiet al.  reagent coupled with Polytron® (Kinematica Inc.) homogenization at room temperature (RT). Afterwards, 200 µl of chloroform was added for every 750 µl of TRIZOL. Samples were shaken vigorously using a vortex for 15 seconds, incubated at room temperature for 5 minutes, and centrifuged at 11000 rpm for 10 minutes at 4°C. The upper aqueous phase was transferred carefully to precipitate the RNA by mixing with 1 volume of isopropanol. Samples were incubated at RT for 15 minutes and centrifuged at 14000 rpm for 10 minutes at 4°C. Next, samples were washed with 80% ethanol in diethyl pyrocarbonate water, and the RNA pellet was air-dried for 5-10 minutes. RNA concentration and quality were measured using a NanoDrop Spectrophotometer . cDNA was synthesized from 100 ng of total RNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s instructions. cDNA samples were stored at −20°C.-Relative quantification by Real-Time Polymerase Chain Reaction (qRT-PCR)Before quantification of the gene of interest, selection of an endogenous gene for normalization was performed by construction of standard curves. In this stage, a total of 100 ng cDNA was taken from 6 OSCC cases and 6 controls to verify that the endogenous gene had no variation between study groups. Five serial dilutions of each cDNA were made to generate a concentration range from 25 ng to 200 ng. The qRT-PCR reactions to evaluate the 18S RNAr, B2M, GAPDH and HPRT1 endogenous genes were performed in a final volume of 10 µL, 1X TaqMan Universal PCR Master mix, 1X TaqMan Gene Expression Assay primer and probe mix (Applied Biosystems). The qPCR reactions were performed on a StepOne Plus Real-Time PCR System  according to the manufacturer´s instructions. Standard curves were compared using Student’s t-test. The gene 18S RNAr was selected because it had the lowest significance.The relative mRNA expression levels of E-cadherin were determined using a TaqMan® Gene Expression Assay . The TaqMan probe corresponded to the cdh1/E-cadherin gene joining at exons 2 and 3 from the 284 bp position and amplified a fragment of 61 bp. During the quantification of E-cadherin gene expression using the qRT-PCR assay, 20 ng of cDNA from each patient sample was used in duplicate for each PCR (according to the mixing conditions previously described). The Cq values were normalized to those of the 18S-rRNA endogenous gene, and gene expression was calculated as the fold-change relative to the average expression of the controls using the 2-ΔΔCt method.-Immunohistochemistry (IHC)To confirm the protein presence in the tumoral tissue, immunohistochemical technique was performed. . Tissue samples were processed and evaluated. Three-μm-thick sections were cut, placed on poly-L-lysine-coated slides, deparaffinized in a 60°C oven for 30 minutes and incubated in xylol for 5 minutes. The sections were then rehydrated in decreasing alcohol concentrations  and washed in distilled water. To retrieve the epitopes, the tissue sections were incubated in 10 mM sodium citrate solution, pH 9, in a microwave oven at a maximum power of 750 W for two cycles of 5 minutes each. The sections were then cooled to room temperature and washed with distilled water. The endogenous peroxidases were blocked with 0.9% hydrogen peroxide, and the samples were washed again with distilled water and phosphate-buffered saline solution . The sections were incubated for 30 minutes with primary antibodies against E-cadherin . Next, the sections were incubated with a biotinylated anti-mouse/anti-rabbit secondary antibody and a streptavidin/peroxidase complex  for 30 minutes each, and the reaction products were detected with 3,3′-diaminobenzidine-H2O2 (Dako Corp.).For microarray analysis, negative and positive controls were obtained from paraffin blocks containing samples of healthy mucosa and fibroma using a 5-mm punch and were placed on slides along with the samples.The immunopositivity and immunolocalization were analyzed by an oral pathologist. The E-cadherin membranal protein (mP) was expressed on the cell membrane in all tumor tissues, regardless of the high or low expression status.-Statistical AnalysisComparisons were made between OSCC cases and controls by Student’s t-test. To perform Chi-square or Fisher’s exact test, the conversion of categorical variables was necessary; zero was considered to be the cutoff point for under- or overexpression. Comparisons between mRNA E-cadherin expression levels, mP E-cadherin and disease characteristics, such as TNM and TNM clinical stage, and prospective data were calculated by the Mann-Whitney U-test and Chi square. Statistical analysis was performed using SPSS statistics v23 software, and ≤ 0.05 was considered to be statistically significant.p-values are shown in Sociodemographic and risk factor profiles from the OSCC case and control groups were obtained. Both groups were similar in regard to gender and age distribution as well as smoking status, whereas alcohol consumption was higher in the control group. With the purpose of assessing the influence of risk factors on E-cadherin gene expression, a comparative analysis between E-cadherin mRNA levels and alcohol or tobacco consumption was made in both case and control groups, without showing significant differences   and invasion of lymph nodes (p = .019 rho = .268) but, strikingky, the correlation becomes weakly negative for the global clinical stage (p = .001 rho = -.389), where the trend line shows that as the clinical stage increases, the mRNA levels decrease.The difference in gene expression of E-cadherin between OSCC cases (-4.3 ± 5.6) and controls (-0.3 ± 4.7) was significant p< 0.00 Fig. 1AAp< 0.001Prospective data  were also not significantly related to E-cadherin mRNA levels Fig. A-D, althThe quantitative expression of E-cadherin was dichotomized in high and low expression groups using a value of 0 as the cutoff and was used to generate survival analysis curves. The average survival in the high expression group was 48.7 ± 3.3 months, and survival was reduced to 44.8 ± 3.3 months in the low expression group, although this difference was not significant. However, it can be seen in the survival curves that deaths occurred earlier in patients with low expression of E-cadherin Fig. E.p=.03), as tissues with low mP E-cadherin expression mostly had low levels of E-cadherin mRNA, and conversely tissues with high mP E-cadherin expressed high levels of E-cadherin mRNA , N (p= .12), TNM clinical stage (p= .26) and recurrence (p= .86), persistence (p= .85), therapy response (p= .48) and death (p= .52).E-cadherin at the cell membrane (mP E-cadherin) was marked by immunohistochemistry Fig. A,B. UsinE-cadherin downregulation at the mRNA level occurs due to several mechanisms and plays a key role in the cell motility that is required for the invasion and metastasis cancer hallmark to occur .et al. found that the expression of E-cadherin mRNA in the invasive tumor front was lower than in the central region , which constitutes another possible limitation of these studies.Future studies with larger sample sizes will be needed to ascertain whether the E-cadherin mRNA level could eventually constitute a predictor of lymph node metastasis in OSCC. Such studies would also allow us to confirm our results regarding the non-significant correlations of E-cadherin mRNA levels with prospective variables such as persistence, recurrence and therapy response.in vitro. These E-cadherin regulatory mechanisms include the SNAIL superfamily (Snail 1 and Snail 2) (in vitro)  , SIP1 (3Snail 2) , the micSnail 2) , proteinn vitro) -35, as wn vitro) .Likewise, gene expression is also regulated epigenetically, regulating gene expression through changes in the structure of chromatin that results from the transfer of methyl groups.Several authors have chosen to study concomitantly the expression of E-cadherin with some of the regulatory proteins in situ ,37; thusIn conclusion, our study confirms the importance of gene expression E-cadherin levels in OSCC and shows that its downregulation at the mRNA level is likely to be an early and constant event in the evolution of this disease. Further studies will be necessary to establish whether E-cadherin levels continue to decrease significantly as OSCC progresses.


Overcoming P-glycoprotein (P-gp) efflux is a strategy to improve the absorption and pharmacokinetics of its substrate drugs. Berberine inhibits P-gp and thereby increases the bioavailability of the P-gp substrate digoxin in rodents. However, the effects of berberine on P-gp in chickens are still unclear. Here, we studied the role of berberine in modulating broilers P-gp expression and function through both in situ and in vitro models. In addition, molecular docking was applied to analyze the interactions of berberine with P-gp as well as with chicken xenobiotic receptor (CXR). The results showed that the mRNA expression levels of chicken P-gp and CXR decreased in the ileum following exposure to berberine. The absorption rate constant of rhodamine 123 increased after berberine treatment, as detected using an in situ single-pass intestinal perfusion model. Efflux ratios of P-gp substrates  decreased and the apparent permeability coefficients increased after co-incubation with berberine in MDCK-chAbcb1 cell models. Bidirectional assay results showed that berberine could be transported by chicken P-gp with a transport ratio of 4.20, and this was attenuated by verapamil (an inhibitor of P-gp), which resulted in a ratio of 1.13. Molecular docking revealed that berberine could form favorable interactions with the binding pockets of both CXR and P-gp, with docking scores of −7.8 and −9.5 kcal/mol, respectively. These results indicate that berberine is a substrate of chicken P-gp and down-regulates P-gp expression in chicken tissues, thereby increasing the absorption of P-gp substrates. Our findings suggest that berberine increases the bioavailability of other drugs and that drug-drug interactions should be considered when it is co-administered with other P-gp substrates with narrow therapeutic windows. Berberine is a yellow isoquinoline alkaloid isolated from many plant families . It has Abcb1 gene, belongs to the family of adenosine triphosphate (ATP)-binding cassette transporters and is usually localized in excretory and barrier-function tissues, such as the kidney and intestine [P-gp, encoded by the ntestine ,7. P-gp ntestine ,9,10, inntestine . TherefoBerberine dose-dependently increases the bioavailability of digoxin and cyclosporine A, two well-known P-gp substrates, by inhibiting intestinal P-gp . In contThis study aimed to determine the effects of berberine on chicken P-gp expression and functional activity. Our results indicate that berberine is a substrate of chicken P-gp and could down-regulate the expression and efflux activity of chicken P-gp, thus increasing the absorption of P-gp substrates, as verified by in situ and in vitro experiments. These findings are informative for the rational use of drugs in the poultry industry to increase bioavailability or avoid adverse effects; accordingly, this study has practical applications for therapeutic efficacy and food safety.Abcb1 and chicken xenobiotic receptor (CXR) were evaluated in the ileum of broilers by real-time RT-PCR after berberine administration at various concentrations (40 and 80 mg/kg) for 24 h . In addition, the decreased mRNA levels of Abcb1 were in line with the decreased CXR mRNA levels.The mRNA expression levels of for 24 h . CompareAbcb1 mRNA expression by berberine is accompanied by a weaker transport function in the small intestine. The jejunum perfusion was evaluated by monitoring the Rho123 concentration (control and berberine-treated samples) over time, as depicted in Ka and Papp values for Rho123 in the jejunum of broilers are presented in p < 0.05) in Rho123 concentrations in the perfusion fluid (Ka) (p < 0.05) (A perfusion model in the jejunum of broilers was chosen for the drug–drug interaction study (berberine–substrate) to further evaluate whether down-regulated on fluid  and a si < 0.05) .p < 0.05), and 1.92 (p < 0.01), with inhibition rates of 41.48%, 53.3%, and 73.6%, respectively. Extending the berberine treatment time to 8 h did not result in further decreases in the ERs and inhibition rates, indicating that the inhibitory effect of berberine on chicken P-gp depends on the concentration but not the treatment time. Therefore, 2 h of incubation was selected for further analyses of verapamil. Similarly, in verapamil-treated cells, there was no significant difference in AP→BL transported Rho123 (p < 0.05), 1.94 (p < 0.05), and 1.37 (p < 0.01), compared with the ER (6.24) of the control. The rates of inhibition increased to 43.75%, 68.91%, and 78.04%, respectively.We investigated the effect of berberine on P-gp function in MDCK-chAbcb1 cells with Rho123 as a probe substrate. For this, a monolayer of MDCK-chAbcb1 cells was pretreated with berberine at various concentrations  for 2 or 8 h. There was no significant difference in Apical to Basolateral (AP→BL) transported Rho123 among berberine concentrations A,C. Howed Rho123 A. Howeved Rho123 B. The ERd Rho123 . High cop < 0.01) in a concentration-dependent manner compared with the ER of the control  by berberine; the ER values for 5 and 20 μM berberine were 4.70 and 3.79, respectively, compared with 5.67 in the absence of berberine (The transport of clindamycin was significantly inhibited (erberine C. When tp < 0.05). Increasing the berberine concentration to 20 μM did not obviously alter the ER. However, when the berberine concentration was increased to 40 μM, the ER decreased significantly to 1.93 (p < 0.05).For sulfadiazine, berberine produced a biphasic response to P-gp-mediated transport, i.e., stimulation at low concentrations and inhibition at high concentrations D. Under p < 0.05).The transport of ampicillin or enrofloxacin in MDCK-chAbcb1 cells was not substantially influenced by berberine E,F. CompPapp for all of the test drugs in MDCK cells  to an extent. Furthermore, there were no significant changes in the ER and CK cells . These rTo further determine how berberine affects the function of P-gp, we hypothesized that berberine is a substrate of P-gp. To evaluate this hypothesis, a bidirectional transport assay of berberine was performed using MDCK and MDCK-chAbcb1 cells. In MDCK parental cells, the berberine transport ratio was 1.24 A. HoweveThe initial three-dimensional structure of CP-gp was obtained using the homology modeling software ITASSER . The modThe previously reported homology model of CXR  was usedThe docking pose of berberine A and theFor the comparative analysis, the docking of all small molecules was evaluated against two other efflux pumps in chickens, CABCC7 and CMATE transporters . It is aBerberine effectively controls coccidial infection and Clostridium perfringens-associated necrotic enteritis in broilers ,19,20. IAbcb1 in chickens. Though the protein level of P-gp was not analyzed owing to a lack of antibodies to chicken P-gp, the decreased efflux activity of chicken intestinal P-gp was further confirmed by an in situ experiment using Rho123 as a probe substrate. It has been reported that various flavonoids, including berberine, regulate P-gp expression via pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in humans [Abcb1 expression via CXR, as the Abcb1 and CXR genes displayed similar changes in response to berberine. Our results are very similar to the finding that camptothecin  attenuates cytochrome P450 3A4 induction by blocking the activation of human PXR [Abcb1 gene expression through Nrf2-mediated pathway when colitis occurs, however, may through PXR-mediated pathway in a healthy organism. On the other hand, other studies showed that there are species-specific variations in Abcb1 gene regulation. These studies provided evidence implicating complex mechanisms for regulation of the Abcb1 gene [CXR and Abcb1 should be studied further and confirmed in vitro.In this study, we found that berberine down-regulated the expression of n humans . CXR, a n humans . Our resuman PXR . This dicb1 gene ,26,27 Fucb1 gene . We hypocb1 gene ,30. Of cThe inhibitory effect of 40 μM berberine on chicken P-gp was comparable with that of 5000 μM verapamil . This indicates that berberine is a potent P-gp inhibitor. Our results also suggest that berberine is an effective P-gp substrate with a high transport ratio in MDCK-chAbcb1 and a low docking score, in good agreement with previous results in humans . In addiTilmicosin, ciprofloxacin, clindamycin, sulfadiazine, ampicillin, and enrofloxacin are all antimicrobial agents with bacteriostatic activity and are commonly used in the poultry industry. We previously demonstrated that all six drugs are substrates of chicken P-gp . The antHygromycin B, rhodamine 123 (Rho123), verapamil, and berberine were obtained from Aladdin . Tilmicosin, ciprofloxacin, clindamycin, sulfadiazine, ampicillin, and enrofloxacin were kind gifts from the China Institute of Veterinary Drug Control . All other chemicals were of analytical grade and obtained from local suppliers, unless otherwise mentioned.2.The MDCK-chAbcb1 cell line stably expressing chicken P-gp was previously established in our laboratory. Briefly, the full-length chicken cDNA was first cloned into the pcDNA3.1 vector and then transfected into MDCK cells using Lipofectamine 2000. Single cells stably expressing chicken P-gp were collected using the limited dilution method in medium containing hygromycin B (100 μg/mL) to obtain the MDCK-chAbcb1 cell line . The celArbor Acres broilers (one day old) were purchased from the Liuhe Hatchery . The broilers were provided a basal diet and water ad libitum and managed under the recommended humidity and temperature for 6 weeks. Then, 18 healthy broilers with similar body weight (b.w.) were randomly divided into three groups (6 broilers/group). Group I was administered phosphate-buffered saline for 24 h (control). Groups II and III were given 40 and 80 mg/kg b.w. berberine orally, respectively, and treated for 24 h. Chicken treatment procedures were approved by the Science and Technology Agency of Jiangsu Province  and performed in accordance with the guidelines of the Science and Technology Agency of Jiangsu Province and Nanjing Agricultural University.Abcb1 and chicken xenobiotic receptor (CXR) mRNAs were quantified using SYBR Green Realtime PCR Master Mix  on a real-time PCR detection system . GAPDH was used as an internal control. Relative levels of Abcb1 and CXR were analyzed using the 2−ΔΔCt method.Broilers were divided into three experimental groups and drugs were administered as described above. At the end of experiments, animals were killed by decapitation. The ileum samples were collected immediately and stored at −80 °C for further tests. Total RNA was isolated using TRIzol Reagent  according to the manufacturer’s instructions and treated with DNase to remove DNA contamination. RNAs were quantified using a photometer  at 260/280 nm. RNA integrity was confirmed by 1% agarose gel electrophoresis and ethidium bromide staining. Then, cDNAs were synthesized using a kit  according to the manufacturer’s protocol. Chicken The broilers were divided into three experimental groups and drugs were administered as described above. Broilers were fasted for 12 h with free access to water before the perfusion experiment. Then, the animals were anesthetized with urethane intraperitoneally (0.5 mL/kg b.w.) and an incision was made through the abdominal cavity. A 10-cm intestinal segment was carefully exposed to air for intestinal perfusion. Two polyethylene cannulas were inserted through small slits at the proximal and distal ends (inlet and outlet). Initially, the intestinal segment was gently flushed with Krebs-Ringer buffer for about 30 min until a clear effluent flowed out. Then, Rho123 (1.5 ng/mL) solution was introduced into the loop at a constant flow rate of 0.2 mL/min. The intestinal fluid was collected in bottles at the outlet of the intestine at intervals of 10 min for up to 100 min. The lengths and internal radii of the intestinal segments were measured after all of the chickens were killed by cervical dislocation.Papp) and absorption rate constant (Ka) of Rho123 were calculated as follows:Qin/Qout are the intestinal perfusate input and output volumes (mL), Cin and Cout represent the mass concentrations of the enteric importer and exporter perfusate (μg/mL), Q represents the perfusion rate (0.2 mL/min), V is the volume of bowel perfusion, and 2πrl is the area of the mass transfer surface (cm2).All samples were weighed and centrifuged for 10 min at 4500 rpm. The supernatants were filtered using a 0.22-μm filter  and samples were collected for analysis by high-performance liquid chromatography (HPLC). The apparent permeability coefficient  or verapamil  for 2 h or with berberine  for 8 h. Then, the bidirectional transport of Rho123 was carried out by replacing the donor buffer with transport buffer containing the mixture of Rho123 (4 mM) and the indicated concentrations of berberine or verapamil. The samples were collected at 20, 40, 60, and 80 min from the receiving sides and analyzed by HPLC.To confirm whether berberine affects the function of P-gp and the efficiency of inhibition, the bidirectional transport of Rho123  was evaluated using MDCK-chAbcb1 cells. Verapamil (a potent inhibitor of P-gp) served as a positive control. Cells were seeded in a 12-well Transwell plate  at a density of 1.0 × 10To prove that berberine could inhibit the efflux function of P-gp, bidirectional transport experiments were further conducted following the above procedure using commonly used veterinary drugs, including tilmicosin, ciprofloxacin, clindamycin, sulfadiazine, ampicillin, and enrofloxacin, which were identified as potential substrates. The berberine preincubation time  was 2 h and the transport concentrations of these antimicrobial drugs were all 20 μM. Enrofloxacin and ampicillin were transported for 1 h and the others for 4 h. Then, the samples collected from the receiving sides were subjected to an HPLC analysis. Of note, the transport periods for all of the drugs were determined by a preliminary experiment to avoid the saturation of P-gp.To evaluate whether berberine is a substrate for P-gp, bidirectional transport experiments using berberine were performed following the above procedure for Rho123 transport. The preincubation time of verapamil (100 μM) was 2 h and the transport concentration of berberine was 100 μM. After transport for 4 h, the samples collected from the receiving sides were used for HPLC.Papp) and the efflux ratio (ER) were calculated as follows:The apparent permeability coefficients  was calculated by dividing the apically directed translocation (B→A) by the basolaterally directed translocation (A→B) of berberine.Structures of berberine and several known P-gp substrates or inhibitors , were generated from canonical SMILES strings that were obtained from the PubChem database . AvogadrHomology modeling of CXR was performed following a previously described procedure . The homThe docking of all ligands into the active sites of CXR, CP-gp, CABCC7, and CMATE was evaluated using AutoDock Vina  and VegaThe top scoring poses of berberine in the binding pockets of both proteins were selected for molecular dynamics simulations to explore the dynamic nature of protein–berberine interactions. The systems were prepared and simulations were conducted following the methods described above. Both trajectories were analyzed using the “Simulations Interactions Diagram” and “Trajectory Clustering” functions built in Maestro. Cluster representatives from trajectories were re-scored using Adaptive Poisson-Boltzmann Solver (APBS)  built inp < 0.05 was considered significant and p < 0.01 was considered extremely significant.Results are presented as the mean ± standard error of the mean (SEM). SPSS  was used for statistical analyses. One-way analysis of variance and post hoc Tukey’s tests were used to assess the statistical significance of differences. In summary, our work demonstrates that berberine could down-regulate the expression and activity of P-gp in broilers, thus increasing the absorption of P-gp substrates both in situ and in vitro. The current results should be helpful in guiding the rational use of drugs in the poultry industry to increase drug bioavailability or avoid possible adverse effects. Further studies are necessary to assess the impact of berberine on P-gp substrate drugs in broilers to pave the way towards clinical applications.


Phyllomedusa tarsius and the mature peptide was validated by MS/MS sequencing, and was subsequently named phylloseptin-PT. The chemically-synthesized and purified phylloseptin-PT displayed activity against Staphylococcus aureus and Candida albicans. Nevertheless, a range of cationicity-enhanced peptide analogues of phylloseptin-PT, which contained amino acid substitutions at specific sites, exhibited significant increases in antimicrobial activity compared to native phylloseptin-PT. In addition, alternative conformers which were designed and chemically-synthesized with d-lysine, showed potent antimicrobial activity and enhanced bioavailability. These data indicate that phylloseptins may represent potential candidates for next-generation antibiotics. Thus, rational design through modification of natural antimicrobial peptide templates could provide an accelerated path to overcoming obstacles en-route to their possible clinical applications.Antimicrobial peptides belonging to the phylloseptin family are mainly found in phyllomedusine frogs. These peptides not only possess potent antimicrobial activity but exhibit low toxicity against eukaryotic cells. Therefore, they are considered as promising drug candidates for a number of diseases. In a recent study, potent antimicrobial activity was correlated with the conserved structures and cationic amphiphilic characteristics of members of this peptide family. A phylloseptin peptide precursor was discovered here in the skin secretion of  The drug market, in recent centuries, has been dominated by small molecules because of their specific desirable characteristics such as potent pharmacological activity, deterministic pharmacodynamics, and rapid analysis . HoweverPhylloseptins are a family of potent antimicrobial peptides that are widely found in the skin secretions of phyllomedusine frogs . The strPhyllomedusa tarsius skin secretion using a high-throughput method of “shotgun” molecular cloning combined with mass spectrometry. Several cationicity-enhanced peptide analogues were designed, which preserved conserved sequences while enhancing positive charges. Here, one potent antimicrobial peptide analogue was chosen as a template and two synthetic diastereomers, composed of d-amino acid residue substitutions in specific positions, were used to investigate the effect of enhancing the stability of the native peptide. This study describes the isolation of a novel antimicrobial peptide precursor from the Phyllomedusa tarsius skin secretion and the translated peptide precursor was identified as encoding a member of the phylloseptin family. The full-length cDNA was cloned from the skin secretion-derived cDNA library of Phyllomedusa tarsius, employing a degenerate primer  , which was designed according to the regions of highly-conserved peptide transcripts from related phyllomedusine frog species, and a universal primer. The nucleic acid and translated amino acid sequence of the novel phylloseptin-encoding precursor contained an open-reading frame of 66 amino acid residues  and matrix-assisted laser desorption/ionisation-time of flight mass spectrometer (MALDI-TOF/MS). The elution position/retention time of a peptide with a mass corresponding to that predicted for phylloseptin-PT, is indicated in the chromatogram shown in The crude skin secretion of Helical wheel projections of the phylloseptin and its designed analogues are shown below in S. aureus and C. albicans, while the cationicity-enhanced analogues, PS-PT1 and PS-PT2, displayed an enhanced antimicrobial activity of broader spectrum. PS-PT1 showed a 4-fold enhanced inhibition against the three selected organisms. PS-PT2 exhibited dramatically enhanced inhibitory activity against reference organisms, increasing the inhibition by 32-fold, with a range of 8–16 mg/L against C. albicans. However, PS-PT2a, the d-lysine substituted analogue of PS-PT2, nearly lost all antimicrobial activity against reference organisms, only sustaining inhibitory activity against C. albicans. Differing from PS-PT2a, PS-PT2b maintained potent antimicrobial activity, especially showing intense inhibition against Gram-negative E. coli. Nonetheless, no haemolytic effect was detected for all five phylloseptin peptides  by cleavage at the Lys7-Ala8 position and the process of this degradation was sustained for up to 36 h. A small quantity of the 1–16 fragment (FLSLIPKAIKAVGVKA-) was seen after 1 min and persisted to 30 min. Another small quantity of the 3–19 fragment (-LSLIPKAIKAVGVKAKKF-) simultaneously appeared during this period. However, PS-PT2a was relatively stable after 72 h. Compared with the wild-type peptide, only a small amount of the 3–19 fragment (LSLIPKAIKAVGVKAKKF) was observed after 4 min as the main degradation pathway, indicating that endopeptidase was the predominant protease. Small quantities of 2–7 fragments (LSLIPK) were subsequently found after a 4 h incubation and kept increasing until the experiment was terminated. For PS-PT2b, although the l-lysine in the seventh position was substituted by d-lysine, the peptide analogue was still hydrolytically-degraded after a 30 s incubation with trypsin. However, the scissile site was changed to Lys10-Ala11 compared to the original site Lys7-Ala8. This is because trypsin specifically targets l-conformed positively-charge amino acids. Thus, even though the 7-lysine was substituted with a d-enantiomer, other l-conformed positive sites can be cleaved. However, PS-PT2b exhibited strong stability in a serum environment compared with the wild-type peptide PS-PT2. In addition, the fragments, 1–15 (FLSLIPKAIKAVGVK), 1–14 (FLSLIPKAIKAVGV), and 1–17 (FLSLIPKAIKAVGVKAK), were observed only after prolonged incubation (>48 h). The degradation of wild-type PS-PT2 was rapid, both by horse serum and trypsin . The oriPhyllomedusa tarsius. The mature peptide sequence was found to end with a glycine residue, serving as an amide donor in the process of post-translational modification. As the observed mass-to-charge (m/z) ratio of the y2-ion was found to be 1 Da less than calculated, this confirmed the presence of the amide modification at the C-terminus. Interestingly, this mature peptide has been previously reported as PS-13 from the skin secretion of Phyllomedusa hypochondrialis azurea, and has been successfully identified by mass spectrometry without the cDNA encoding sequence [Phyllomedusa hypochondrialis azurea. The precursor of PS-PT is similar to the others except for a typical basic dipeptide, -KR-, in the middle (positions 35 and 36) of the putative acidic “spacer” region, instead of acidic and amidic residues  and Asp (at position 10) with Lys, and was named PS-PT2. This design combined the conserved motif with enhanced cationicity. Additionally, the hydrophobic moment of PS-PT2, an important structural characteristic for α-helical antimicrobial peptides, was higher than the other peptide analogues. This design is suggested to stabilise the amphipathic structure and ensures a proper residue distribution in the hydrophobic and hydrophilic faces in the helical wheel. The antimicrobial activity of this designed analogue was significantly increased by 32-fold against l-lysine by d-lysine may maintain potent antimicrobial activity and provide resistance to protease degradation [d-lysine was used to synthesize an optical isomer using PS-PT2 as a template and this peptide was named PS-PT2a. Incubation of PS-PT2 and PS-PT2a with horse serum and trypsin, respectively, revealed that PS-PT2 was degraded rapidly at the most susceptible site of l-lysine at position 7 in a few minutes, while the d-lysine substituted peptide was barely degraded at all over 48 h. This result indicates that the antimicrobial potency of PS-PT2 might be gone within a few minutes in vivo and this obviously limits its effects, while the antimicrobial activity of those fragments could be further investigated. The antimicrobial assay showed that the antimicrobial activity of PS-PT2a is defective compared to PS-PT2. It could be explained by the fact that the d-amino acids affect the secondary structure, especially the α-helix in this sequence. This has clearly shown that the α-helicity of PS-PT2a declined dramatically compared to PS-PT2, from 37.91% to 1.06%. Meanwhile, it has been proposed that increasing net positive charge of a peptide in a neutral environment is a good approach to enhance antimicrobial activity, but the maintenance of amphipathicity and the helical domain are more significant.Nonetheless, the stability of natural peptides is a major reason for developing novel drug delivery methods, as peptides are easily degraded into small fragments or amino acids by various endogenous enzymes. However, it has been reported that the replacement of radation . In thisd-amino acid. In this study, the l-lysine at position 7 was replaced by its d-isomer and the peptide was named PS-PT2b. This peptide retained its antimicrobial activity and stability in horse serum. The retained activity of PS-PT2b was less potent than PS-PT2, especially against the Gram positive bacterium, S. aureus. It was presumed that Lys-7 had a high propensity for forming an α-helix and that this α-helical structure was required for activity against Gram positive bacteria. The result of the CD has confirmed this, assuming that the α-helicity of PS-PT2b was slightly less than PS-PT2, about 23.92%. The α-helicity sustaining in a certain range is a necessary condition for the maintenance of potent antimicrobial activity. These data offered an insight into a possible strategy for the design of antimicrobial peptides against selected bacteria by the use of d-amino acid substitutions.An alternative method to overcome this limitation is to replace the amino acid in the most protease susceptible site with a Phyllomedusa tarsius frogs were acquired from a commercial source  and the skin secretions were obtained by mild transdermal electrical stimulation [The mulation . FollowiPhyllomedusa bicolor and the opioid peptide cDNA from Phyllomedusa dacnicolor [The isolation of pure mRNA from crude skin secretion was achieved by utilizing a magnetic oligo-dT bead kit  which could bind polyadenylated mRNA in the cell lysis buffer supplied with the kit. Reverse transcription and synthesis of first-strand cDNA was followed by a 3’-RACE reaction to isolate target antimicrobial peptide precursor nucleic acid sequence data with a SMART-RACE kit . 3’-RACE was facilitated by a nested universal primer (NUP) (supplied by the kit) and a sense primer   which was designed according to the highly conserved segment of the 5′-untranslated region of phylloxin cDNA from cnicolor ,23. The v/v) to acetonitrile/water/TFA  on an analytical column . The effluent was constantly monitored by a UV detector set at 214 nm (λ) and the fractions were automatically collected at minute intervals. All fractions were analysed by MALDI-TOF/MS  with CHCA as the matrix in positive mode. The instrument was calibrated by standards and set accuracy was ±0.1%. The peptide with a molecular mass coincident with that predicted from cloned cDNA, was injected into an LCQ-Fleet electrospray ion-trap mass spectrometer to analyse its primary structure by MS/MS fragmentation .Five mg of lyophilised skin secretion were dissolved, clarified, and injected into an HPLC system  to separate the fractions using a gradient programme which ran over 240 min at a flow rate of 1 mL/min from water/TFA . Each peptide was dissolved in 10 mM ammonium acetate and 10 mM ammonium acetate with 50% TFE, respectively and was prepared at 100 μM in a 1 mm high precision quartz cell . CD spectra were recorded at a wavelength ranging from 190 nm to 250 nm with a 100 nm/min scan speed. The parameters were set as 1 nm bandwidth and 0.5 nm data pitch. The result was analysed by the K2D3 webserver to estimate α-helical content . The hell-amino acids, while d-lysine substituted cationicity-improved peptides were synthesized with d-lysine and other l-amino acids in their sequences. The process was carried out by standard Fmoc chemical synthesis, deprotection, and cleavage procedures. Each peptide was purified by HPLC and structurally-validated by mass spectrometry.The natural peptide and respective cationicity-enhanced analogues, were chemically-synthesized by an automatic solid-phase synthesiser  with natural Staphylococcus aureus (NCTC 10788), Escherichia coli (NCTC 10418), and Candida albicans (NCPF 1467). Briefly, the reference strains were incubated in Muller-Hinton Broth (MHB) until logarithmic phase growth was achieved which was assessed by the optical density (OD) of the culture at 550 nm. The cultures were subjected to dilution to obtain 1 × 106 colony-forming units (cfu)/mL for bacteria or to 5 × 105 cfu/ml for the yeast. Serial peptide dilutions and cultures were loaded into a 96-well microtiter plate obtaining suspensions containing 1–512 mg/L tested peptides. After incubating at 37 °C for 24 h, MIC values were determined as the lowest concentration which produced a similar OD value to the negative control (medium only). Then 10 μL of inhibited culture from each well was subcultured on Muller-Hinton Agar (MHA) and incubated for 24 h. The MBC values were defined as the minimum concentration which effected no growth.The antimicrobial activity was evaluated by minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays using broth microdilution methods and in vitro assays against reference strains of microorganisms; a Gram-positive bacterium, a Gram-negative bacterium, and a yeast, respectively, v/v) was formed with prewashed defibrinated horse erythrocytes and sodium phosphate-buffered saline (PBS). Peptides were incubated with this 2% suspension in a final concentration range from 1 to 512 mg/L and all the samples were kept at a constant 37 °C for 120 min. Negative controls and positive controls were PBS alone and PBS containing Triton X-100 , respectively. The sample supernatants were used to assess the extent of haemolysis by measuring the OD value at 550 nm.The haemolytic activity of peptides against defibrinated horse erythrocytes  was determined by comparing the degree of lysis after incubating tested peptides with prewashed red cell suspensions for 2 h. A 2% suspension (v/v) and incubated at 37 °C for 15 min. Peptide stock solution was added into the serum medium to obtain a solution having a final concentration of 1 mg/mL and test samples were kept at 37 °C for 72 h. At different time points, 20 μL samples were transferred into 100 μL of 0.25% (v/v) TFA/water which caused reaction termination. For the serum-digestion, samples were kept at 4 °C and centrifuged at 13,000 rpm for 15 min and the supernatants were then analysed by MALDI-TOF/MS.Two different media were used to appraise the stability and degradation sites of peptides, trypsin, and horse serum, respectively. For the trypsin assay, 1 mg of trypsin (Sigma-Aldrich) with 1 mg of peptide were incubated in 1 mL PBS for 72 h. Whilst for horse serum, the medium was prepared with 25% horse serum/RPMI medium (Phyllomedusa tarsius skin secretion, utilizing a high throughput system combining mass spectrometric sequencing and “shotgun” molecular cloning to obtain the cDNA sequence. Following this, the study progressed by assessing the bioactivity, toxicity, and stability of the peptide. Four cationicity-enhanced peptide analogues were designed, chemically synthesized, and evaluated. The cationicity-enhanced peptides exhibited more potent antimicrobial activity compared to the natural peptide. The data demonstrated that site-selective amino acid substitutions with d-isomers could sustain a similar antimicrobial activity with prolonged stability. Generally, these studies elucidated a method to discover and evaluate novel natural antimicrobial peptides, and to design and synthesise analogues with enhanced potency, specific activity, and stability to potentially develop as novel therapeutic agents.This work describes the isolation and structural elucidation of an antimicrobial peptide from the


Cocaine dependence is a psychiatric condition for which effective medications are still lacking. Published data indicate that an increase in nociceptin/orphanin FQ (N/OFQ) transmission by NOP receptor activation attenuates cocaine-induced place conditioning and the locomotor sensitization effects of cocaine. This suggests that the activation of the N/OFQ receptor (NOP) may attenuate the motivation for psychostimulants. To further explore this possibility, we investigated the effect of the potent and selective NOP receptor agonist Ro 64-6198 on cocaine intake under 1 h short access (ShA) and 6 h long access (LgA) operant self-administration conditions in rats. We used Marchigian Sardinian alcohol-preferring (msP) rats and Wistar control rats. msP rats were used because we recently found that this rat line, originally selected for excessive alcohol drinking and preference, exhibits a greater propensity to escalate cocaine self-administration following LgA training. msP rats are also characterized by innate overexpression of the N/OFQ-NOP system compared with Wistar rats. Wistar and msP rats both exhibited an increase in cocaine self-administration under LgA conditions, with a higher trend toward escalation in msP rats. In Wistar rats, the intraperitoneal administration of Ro 64-6198 (0. 1 and 3 mg/kg) significantly decreased ShA cocaine self-administration. In Wistar rats that underwent LgA cocaine self-administration training, Ro 64-6198 induced no significant effect either during the first hour of self-administration or after the entire 6 h session. In msP rats, Ro 64-6198 significantly reduced cocaine self-administration both under ShA conditions and in the first hour of the LgA session. At the end of the 6 h session, the effect of Ro 64-6198 was no longer observed in msP rats. The highest dose of Ro 64-6198 (3 mg/kg) did not affect saccharin self-administration in msP rats but reduced saccharin self-administration in Wistar rats. Altogether, these data suggest that NOP receptor activation attenuates cocaine self-administration, and this effect tends to be more pronounced in a rat line with innately higher NOP receptor expression and that more robustly escalates cocaine intake. Cocaine is the most commonly abusedillicit psychostimulant, and its use is linked to serious physical, psychiatric, socioeconomic, and legal problems . EffectiCocaine is often co-abused with alcohol. Cocaine dependence and alcohol dependence share several genetic traits, indicating that common predisposing factors may exist –4. We reper se .The experiments were conducted with male Wistar rats  and msP rats . The rats weighed 200–250 g at the beginning of the study. They were housed in pairs in a room under a reverse 12 h/12 h light/dark cycle (lights off at 9:00 a.m.) with constant temperature (20–22°C) and humidity (45–55%). Food and water were provided Cocaine hydrochloride  was dissolved in sterile saline. Saccharin  was dissolved in tap water. The NOP receptor agonist Ro 64-6198 was dissolved in 10% dimethylsulfoxide, 10% Tween-80, and 80% water. Doses timing and route of administration of Ro 64-6198 were chosen based on earlier NOP binding studies indicating that an acute IP injection of 3.2 mg/kg of Ro 64-6198 induced maximal NOP receptor occupancy after 30 min. Good receptor occupancy was maintained for approximately 3 h .The rats were anesthetized by an intramuscular injection of 100–150 μl of a solution that contained tiletamine chlorhydrate (58.17 mg/ml) and zolazepam chlorhydrate (57.5 mg/ml). For intravenous surgery, incisions were made to expose the right jugular vein. A catheter that was constructed from micro-renathane tubing  was subcutaneously positioned between the vein and back. After insertion into the vein, the proximal end of the catheter was anchored to the muscles that underlie the vein with surgical silk. The distal end of the catheter was attached to a stainless-steel cannula that was bent at a 90° angle. The cannula was inserted into a support that consisted of dental cement on the back of the animals and was covered with a plastic cap. Immediately after surgery, the rats were treated intramuscularly with 200 μl of enrofloxacin .The rats were allowed to recover for 1 week before self-administration training. Catheters patency was confirmed by an intravenous injection of 150 μl of sodium pentothal . Before each self-administration session, the catheters were flushed with 100 μl of heparinized saline (20 UI/ml) that contained 0.5 mg/ml enrofloxacin.The self-administration stations consisted of operant conditioning chambers  that were enclosed in sound-attenuating, ventilated environmental cubicles. Each chamber was equipped with two retractable levers that were located in the front panel of the chamber. Cocaine was delivered intravenously through a plastic tube that was connected to an infusion pump. Saccharin was delivered in a receptacle that was connected to the infusion pump and located between the two levers. Responses on the right (active) lever activated the infusion pump, and responses on the left (inactive) lever were recorded but did not have any programmed consequences. In both the cocaine and saccharin sessions, activation of the pump resulted in the delivery of 0.1 ml of fluid. A computer controlled the delivery of cocaine solution and recorded the behavioral data.Wistar and msP rats were initially trained to self-administer cocaine (0.25 mg/infusion) under a fixed-ratio 1 (FR1) schedule of reinforcement for 1 h/day for 5 days. Afterward, the reinforcement contingency was switched to an FR5 schedule, and the rats of each line were split into two subgroups: self-administration in a 1 h short-access (ShA) session and self-administration in a 6 h long access (LgA) session. The LgA and ShA sessions under an FR5 schedule continued for 23 days, after which the effect of Ro 64-6198 on cocaine self-administration was tested.n = 10) and LgA (n = 10) conditions. Using a within-subjects counterbalanced design, 30 min before the session, the rats received Ro 64-6198  or its vehicle. The tests were repeated every third day. On the first intervening day, the rats remained in their home cage. On the second intervening day, they underwent a baseline cocaine self-administration session. The number of infusions was recorded after the first hour of self-administration and also at 6 h in the LgA group.Starting from the 24th FR5 session, we tested the effect of Ro 64-6198 and its vehicle on cocaine self-administration in Wistar rats that were trained under ShA (n = 9) and LgA (n = 9) conditions. The tests were performed using a within-subjects counterbalanced design at intervals of 3 days. The number of infusions was recorded after the first hour of self-administration and also at 6 h in the LgA group.Similar to Wistar rats, starting from the 24th FR5 session, we tested the effect of Ro 64-6198  and its vehicle on cocaine self-administration in msP rats that were trained under ShA (n = 9) and msP (n = 9) rats were trained in daily 1 h saccharin self-administration sessions under an FR1 schedule of reinforcement. When the rats reached a stable baseline of saccharin intake, we tested the effect of Ro 64-6198  and its vehicle on saccharin self-administration. Thirty minutes before the sessions, the rats received Ro 64-6198  and its vehicle in a counterbalanced Latin-square design. The tests were repeated every third day.Two additional groups of Wistar (vs. LgA) as the between-subjects factor and time (days) as the repeated measure. The escalation of cocaine self-administration was analyzed using one-way ANOVA, with time as the repeated measure. The effect of Ro 64-6198 on cocaine and saccharin self-administration was analyzed using one-way ANOVA, with dose as the within-subjects factor. Wistar and msP rats were analyzed separately. Significant main effects in the ANOVA were followed by the Newman-Keuls post hoc test for escalation and Dunnett's post hoc test for Ro 64-6198. Values of p < 0.05 were considered statistically significant.The number of infusions that were received by ShA rats was compared with the number of infusions that were received by LgA rats during the first hour of the daily sessions using two-way repeated-measures analysis of variance (ANOVA), with session length  = 1.5, p > 0.05] but a significant effect of time  and a significant session length × time interaction . The two groups differed in intake on the first day under the FR5 schedule but earned a similar number of infusions during the remainder of training  = 8.1, p < 0.01], with a progressive increase in the total number of daily (6 h) cocaine infusions that were earned  = 40.9, p < 0.0001] and time  and a significant session length × time interaction . The post hoc analysis indicated that LgA rats exhibited an increase in the number of infusions over time and differed significantly from ShA rats starting from the fifth session  = 8.9, p < 0.001], with a progressive increase in the total number of daily (6 h) cocaine infusions that were earned  = 16.5, p < 0.01]. Dunnett's post hoc test revealed that 3.0 mg/kg but not 1.0 mg/kg Ro 64-6198 significantly decreased the number of infusions compared with vehicle  = 2.8, p > 0.05] or during the entire 6 h session . Dunnett's post hoc test showed that 3 mg/kg Ro 64-6198 significantly decreased saccharin self-administration  = 15.3, p < 0.001; F = 11.6, p < 0.01; post hoc test revealed a significant effect of 3.0 mg/kg Ro 64-6198 (p < 0.01). The ANOVA of the effect of Ro 64-6198 at 6 h in the LgA group revealed no effect of treatment [F = 0.3, p > 0.05; F = 2.0, p > 0.05; In msP rats, Ro 64-6198 significantly decreased cocaine self-administration both in the ShA condition [The present study found that cocaine intake was similar in Wistar and msP rats under ShA self-administration conditions, but Wistar rats received a slightly higher number of infusions. When the rats were exposed to LgA self-administration, msP rats exhibited greater escalation of cocaine intake. These results replicate our recent study, in which we found that msP rats exhibited greater escalation of cocaine self-administration compared with Wistar rats. Additionally, in response to an amphetamine challenge, msP rats exhibited greater locomotor stimulation, greater activation of mesolimbic circuitry, and higher extracellular levels of dopamine in the nucleus accumbens compared with Wistar rats . The msP1 and D2 receptors in the striatum  and acute treatment with Ro64-6198 produced comparable reduction of NOP binding levels due to receptor internalization . Based oIn conclusion, the present study found that msP rats escalated their cocaine self-administration more rapidly than Wistar rats, and NOP receptor activation by Ro 64-6198 reduced cocaine consumption. The mechanism by which NOP receptor agonism leads to such effects is unknown, but receptor desensitization may be one possibility.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.RC was responsible for the study concept and design. HL, GS, and QS performed the experiments. CN and AM assisted with the data analysis and interpretation of findings. HL, AM, NC, and RC drafted the manuscript. All of the authors critically reviewed the content and approved the final version for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.